Green fluorescent protein

ABSTRACT

This invention provides a cell comprising a DNA molecule having a regulatory element from a gene, other than a gene encoding a green fluorescent protein operatively linked to a DNA sequence encoding the green fluorescent protein. This invention also provides living organisms which comprise the above-described cell. This invention also provides a method for selecting cells expressing a protein of interest which comprises: a) introducing into the cells a DNAI molecule having DNA sequence encoding the protein of interest and DNAII molecule having DNA sequence encoding a green fluorescent protein; b) culturing the introduced cells under conditions permitting expression of the green fluorescent protein and the protein of interest; and c) selecting the cultured cells which express green fluorescent protein, thereby selecting cells expressing the protein of interest. Finally, this invention provides various uses of a green fluorescent protein.

The invention disclosed herein was made with Government support underNIH Grant No. 5R01GM30997 from the Department of Health and HumanServices. Accordingly, the U.S. Government has certain rights in thisinvention.

This application is a continuation-in-part of U.S. Ser. Nos. 08/119,678,U.S. Pat. No. 5,491,084, and 08/192,274, abandoned, InternationalApplicaton No. PCT/US94/10165 filed Sept. 10, 1993 and Feb. 4, 1994,Sep. 9, 1994 respectively, the contents of which are hereby incorporatedby reference.

BACKGROUND OF THE INVENTION

Throughout this application various references are referred to withinparenthesis. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citation for these references may be found at the end ofthis application, preceding the sequence listing and the claims.

Several methods are available to monitor gene activity and proteindistribution within cells. These include the formation of fusionproteins with coding sequences for β-galactosidase (22), and luciferases(22). The usefulness of these methods is often limited by therequirement to fix cell preparations or to add exogenous substrates orcofactors. This invention disclose a method of examining gene expressionand protein localization in living cells that requires noexogenously-added compounds.

This method uses a cDNA encoding the Green fluorescent Protein (GFP)from the jelly fish Aequorea victoria (3). In A. victoria, GFP absorbsenergy generated by aequorin upon the stimulation by calcium and emits agreen light.

This invention discloses that GFP expressed in prokaryotic andeukaryotic cells is capable of producing a strong green fluorescencewhen excited with near UV or blue light. Since this fluorescencerequires no additional gene products from A. victoria, chromophoreformation is not species specific.

SUMMARY OF THE INVENTION

This invention provides a cell comprising a DNA molecule having aregulatory element from a gene, other than a gene encoding a greenfluorescent protein operatively linked to a DNA sequence encoding thegreen fluorescent protein. This invention also provides living organismscomprising the above-described cell.

This invention provides a method for selecting cells expressing aprotein of interest which comprises: a) introducing into the cells aDNAI molecule having DNA sequence encoding the protein of interest andDNAII molecule having DNA sequence encoding a green fluorescent protein;b) culturing the introduced cells in conditions permitting expression ofthe green fluorescent protein and the protein of interest; and c)selecting the cultured cells which express green fluorescent protein,thereby selecting cells expressing the protein of interest.

This invention also provides a method for localizing a protein ofinterest in a cell: a) introducing into a cell a DNA molecule having DNAsequence encoding the protein of interest linked to DNA sequenceencoding the green fluorescent protein such that the protein produced bythe DNA molecule will have the protein of interest fused to the greenfluorescent protein; b) culturing the cell in conditions permittingexpression of the fused protein; c) detecting the location of the fusedprotein product, thereby localizing the protein of interest.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 Expression of GFP in E. coli. The bacteria on the right side ofthe figure have the GFP expression plasmid. This photograph was takenwhile irradiating the agar plate with a hand-held long-wave UV source.

FIG. 2 Excitation and Emission Spectra of E. coli-generated GFP (solidlines) and purified A. victoria GFP (L form; dotted lines).

FIG. 3 Expression of GFP in a first stage Caenorhabditis elegans larva.Two touch receptor neurons (PLML and ALML) and one other neuron ofunknown function (ALNL) are indicated. Processes can be seen projectingfrom all three cell bodies. The arrow points to the nerve ring branchfrom the ALML cell (out of focus). The background fluorescence is due tothe animal's autofluorescence.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this application, the following standard abbreviations areused to indicate specific nucleotides:

    ______________________________________                                        C = cytosine         A = adenosine                                            T = thymidine        G = guanosine                                            ______________________________________                                    

This invention provides a cell comprising a DNA molecule having aregulatory element from a gene, other than a gene encoding a greenfluorescent protein operatively linked to a DNA sequence encoding thegreen fluorescent protein.

This invention provides a cell comprising a DNA molecule having aregulatory element from a gene, other than a gene encoding a greenfluorescent protein operatively linked to a DNA sequence encoding thegreen fluorescent protein, wherein the cell is selected from a groupconsisting essentially of bacterial cell, yeast cell, fungal cell,insect cell, nematode cell, plant or animal cell.

Suitable animal cells include, but are not limited to Vero cells, HeLacells, Cos cells, CVI cells and various vertebral, invertebral,mammalian cells.

In an embodiment, the bacterial cell is Escherichia coli. As usedherein, "a regulatory element" from a gene is the DNA sequence which isnecessary for the transcription of the gene.

In this invention, the term "operatively linked" means that followingsuch a link the regulatory element can direct the transcription of thelinked protein-coding DNA sequence.

The gene encoding a green fluorescent protein includes DNA moleculescoding for polypeptide analogs, fragments or derivatives of antigenicpolypeptides which differ from naturally-occurring forms in terms of theidentity or location of one or more amino acid residues (deletionanalogs containing less than all of the residues specified for theprotein, substitution analogs wherein one or more residues specified arereplaced by other residues and addition analogs where in one or moreamino acid residues is added to a terminal or medial portion of thepolypeptides) and which share some or all properties ofnaturally-occurring forms.

These DNA molecules include: the incorporation of codons "preferred" forexpression by selected mammalian or non-mammalian hosts; the provisionof sites for cleavage by restriction endonuclease enzymes; and theprovision of additional initial, terminal or intermediate DNA sequencesthat facilitate construction of expression vectors.

As an example, plasmid pGFP10.1 codes for a mutated GFP protein havingthe 80th amino acid residue as an arginine rather than a glutaminepredicted to be in native GFP from A. Victoria. This mutated proteinretains the property to fluoresce like the natural protein.

In an embodiment, the regulatory element is a promoter. In a furtherembodiment, the promoter is activated by a heavy metal. Such promotersare well-known in the art (J. H. Freedman, L. W. Slice, A. Fire, and C.S. Rubin (1993) Journal of Biological Chemistry, 268:2554).

In another embodiment, the promoter is that from a cytochrome P450 gene.Cytochrome P450 is well-known in the art and there are a number of P450promoters known.

In still another embodiment, the promoter is that from a stress proteingene. Such stress proteins are well-known in the art (E. G. Stringham,D. K. Dixon, D. Jones and E. D. Candido (1992) Molecular Biology of theCell, 3:221; and William J. Welch (May, 1993), Scientific American, page56). In a further embodiment, the stress protein is a heat-shockprotein.

This invention provides a cell comprising a DNA molecule having aregulatory element from a gene, other than a gene encoding a greenfluorescent protein operatively linked to a DNA sequence encoding thegreen fluorescent protein, wherein the promoter is from a gene necessaryfor the viability of a cell.

In another embodiment, the regulatory element is an enhancer. Enhancersare well-known in the art.

This invention provides a cell comprising a DNA molecule having aregulatory element from a gene, other than a gene encoding a greenfluorescent protein operatively linked to a DNA sequence encoding thegreen fluorescent protein, wherein the DNA sequence encodes the Aequoreavictoria green fluorescent protein.

In an embodiment, the Aequorea victoria green fluorescent protein iscloned in a plasmid. This plasmid is a modification of the pBS(+)(formerly called Bluescribe+) vector (Stratagene®) which has insertedwithin it an EcoRI fragment containing the cDNA sequence of the AequoreaVictoria green fluorescent protein (as modified herein). The fragmentwas obtained from λFP10 (Prasher, D. C., Eckenrode, V. K., Ward, W. W.;Prendergast, F. G., and Cormier, M. J., (1992) Primary structure of theAequorea victoria green fluorescent protein. Gene, 111:229-233) byamplification using the polymerase chain reaction (Saiki, R. K.,Gelfand, D. H., Stoffel, S., Sharf, S. J., Higuchi, G. T., Horn, G. T.,Mullis, K. B., and Erlich, H. A. (1988) Primer-directed enzymaticamplification of DNA with a thermostable DNA polymerase. Science,239:487-491) with primers flanking the EcoRI sites and subsequentdigestion with EcoRI. The sequence of the cDNA in pGFP10.1 differs fromthe published sequence (5) by a change of the 80th codon of the codingsequence from CAG to CGG, a change that replaces a glutamine witharginine in the protein sequence.

This pGFP10.1 plasmid was deposited on Sep. 1, 1993 with the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va. 20110, U.S.A. under the provisions of the Budapest Treaty for theInternational Recognition of the Deposit of Microorganism for thePurposes of Patent Procedure. Plasmid pGFP10.1 was accorded ATCCAccession Number 75547.

In another embodiment, this invention provide a bacterial cell which isexpressing the green fluorescent protein. In an further embodiment, thebacterial cell is an E. coli cell. In a still further embodiment, thisE. coli cell is designated SMC1 (ATCC Accession No. 69554).

This SMC1 bacterial cell was deposited on Feb. 4, 1994, 1993 with theAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110, U.S.A. under the provisions of the Budapest Treatyfor the International Recognition of the Deposit of Microorganism forthe Purposes of Patent Procedure. Bacterial cell SMC1 was accorded ATCCAccession Number 69554.

This invention further provides an isolated green fluorescent proteinproduced from the above-described cells which comprise a DNA moleculehaving a regulatory element from a gene, other than a gene encoding agreen fluorescent protein operatively linked to a DNA sequence encodingthe green fluorescent protein. This isolated green fluorescent proteincan then be further modified in vitro for various uses.

This invention disclose an efficient method for expression of greenfluorescent protein such that large amount of the protein could beproduced. Methods to isolate expressed protein have been well-known andtherefore, green fluorescent protein may be isolated easily.

This invention provides a living organism comprising the cell comprisinga DNA molecule having a regulatory element from a gene, other than agene encoding a green fluorescent protein operatively linked to a DNAsequence encoding the green fluorescent protein.

In another embodiment, the living organism is human. In anotherembodiment, the living organism is a mouse. The living organism may beother mammals. In addition, this invention is applicable to othervertebrates, non-vertebrates and living organisms.

In an embodiment, the living organism is C. elegans. In still anotherembodiment, the living organism is Drosophila, zebra fish, virus orbacteriophage.

A bacteriophage carrying the green fluorescent protein gene can infect aparticular type of bacteria. The infection may be easily detected viathe expression of the green fluorescent protein. Therefore, by usingappropriate bacteriophages, the presence of that particular type ofbacteria may be detected.

Similarly, a eucaryotic virus carrying the green fluorescent proteingene may infect a specific cell type. The infection may be easilydetected by monitoring the expression of the green fluorescent protein.

Methods to introduce exogenous genetic material into a cell arewell-known in the art. For example, exogenous DNA material may beintroduced into the cell by calcium phosphate precipitation technology.Other technologies, such as the retroviral vector technology,electroporation, lipofection and other viral vector systems such asadeno-associated virus system, or microinjection may be used.

The above-described cells and living organisms are useful to detecteffects of external stimulus to the regulatory element. The stimulus mayhave direct or indirect effects on the regulatory element. Such effectswill be detectable through either the induction of expression andproduction of the green fluorescent protein or switching off theexpression of the green fluorescent protein.

Cells expressing the green fluorescent proteins may be convenientlyseparated by a fluorescence-activated cell sorter.

These cells and organisms may be used to detect the presence ofdifferent molecules in various kinds of biological samples such asblood, urine or saliva. By operatively linking a regulatory element ofthe gene which is affected by the molecule of interest to a greenfluorescent protein, the presence of the molecules will affect theregulatory element which in turn will affect the expression of the greenfluorescent protein. Therefore, the above-described cells are useful forthe detection of molecules. Such detection may be used for diagnosticpurposes. An example of such a molecule is a hormone.

This invention provides a living organism comprising the cell comprisinga DNA molecule having a regulatory element from a gene, other than agene encoding a green fluorescent protein operatively linked to a DNAsequence encoding the green fluorescent protein, wherein the regulatoryelement is for a stress protein.

This invention provides a living organism comprising the cell comprisinga DNA molecule having a regulatory element from a gene, other than agene encoding a green fluorescent protein operatively linked to a DNAsequence encoding the green fluorescent protein, wherein the stressprotein is a heat-shock protein.

This invention provides a method to produce green fluorescent proteincomprising a) culturing the above-described cells comprising a DNAmolecule having a regulatory element from a gene, other than a geneencoding a green fluorescent protein operatively linked to a DNAsequence encoding the green fluorescent protein; and b) isolating andpurifying the green fluorescent protein produced from the cells.Standard methods for isolating and purifying proteins are well-known inthe art. In an embodiment, the cells used for production of greenfluorescent proteins are E. coli cells. In a further embodiment, the E.coli cells are cultured aerobically.

This invention provides a method to synthesize green fluorescent proteincomprising a) culturing the cell designated SMCl; and b) isolating andpurifying the green fluorescent protein produced from the cell.

This invention provides a method for selecting cells expressing aprotein of interest which comprises: a) introducing into the cells aDNAI molecule having DNA sequence encoding the protein of interest andDNAII molecule having DNA sequence encoding a green fluorescent protein;b) culturing the introduced cells in conditions permitting expression ofthe green fluorescent protein and the protein of interest; and c)selecting the cultured cells which express green fluorescent protein,thereby selecting cells expressing the protein of interest.

This invention also provides the above method, wherein the cells areselected from a group consisting essentially of bacterial cells, yeastcells, fungal cells, insect cells, nematode cells, plant or animalcells. Suitable animal cells include, but are not limited to Vero cells,HeLa cells, Cos cells, CV1 cells and various primary mammalian cells.

In an embodiment, DNAI and DNAII are linked. In another embodiment, theDNA encodes the Aequorea Victoria green fluorescent protein.

This invention provides a method for localizing a protein of interest ina cell which comprises: a) introducing into a cell a DNA molecule havingDNA sequence encoding the protein of interest linked to DNA sequenceencoding a green fluorescent protein such that the protein produced bythe DNA molecule will have the protein of interest fused to the greenfluorescent protein; b) culturing the cell in conditions permittingexpression of the fused protein; and c) detecting the fused proteincomposed of the green fluorescent protein in the cell, therebylocalizing a protein of interest in a cell.

Regulatory elements required for expression include promoter sequencesto bind RNA polymerase and translation initiation sequences for ribosomebinding. For example, a bacterial expression vector includes a promotersuch as the lac promoter and for translation initiation theShine-Dalgarno sequence and the start codon ATG. Similarly, a eukaryoticexpression vector includes a heterologous or homologous promoter for RNApolymerase II, a downstream polyadenylation signal, the start codon ATG,and a termination codon for detachment of the ribosome. Such vectors maybe obtained commercially or assembled from the sequences described bymethods well-known in the art, for example the methods described abovefor constructing vectors in general.

To maximize the expression of the green fluorescent protein, thesequence flanking the translation initiation codon may be modified(reviewed by Kozak, 1984), compilation and analysis of sequencesupstream from the translation start site in eucaryotic mRNAs. Nucl.Acids. Res. 12:857-872). A sequence may then be generated to producehigher amounts of the GFP protein.

In addition, artificial introns may be introduced so as to increase theproduction of the protein.

Other special targeting sequences may be inserted into the GFP gene. Onesuch targeting sequence is the nuclear localization signal (such as theSV40 nuclear localization signal).

The host cell of the above expression system may be selected from thegroup consisting of the cells where the protein of interest is normallyexpressed, or foreign cells such as bacterial cells (such as E. coli),yeast cells, fungal cells, insect cells (such as Sf9 cell in thebaculovirus expression system), nematode cells, plant or animal cells,where the protein of interest is not normally expressed. Suitable animalcells include, but are not limited to Vero cells, HeLa cells, Cos cells,CV1 cells and various primary mammalian cells.

In an embodiment of the method for localizing a protein of interest in acell, the DNA encoding the green fluorescent protein is from Aequoreavictoria.

This invention provides a method for localizing a protein of interest ina cell which comprises: a) introducing into a cell a DNA molecule havingDNA sequence encoding the protein of interest linked to DNA sequenceencoding a green fluorescent protein such that the protein produced bythe DNA molecule will have the protein of interest fused to the greenfluorescent protein; b) culturing the cell in conditions permittingexpression of the fused protein; and c) detecting the location of thefused protein composed of green fluorescent protein in the cell, therebylocalizing a protein of interest in a cell, wherein the cell normallyexpressing the protein of interest.

This invention provides a method for detecting expression of a gene in acell which comprises: a) introducing into the cell a DNA molecule havingDNA sequence of the gene linked to DNA sequence encoding a greenfluorescent protein such that the regulatory element of the gene willcontrol expression of the green fluorescent protein; b) culturing thecell in conditions permitting expression of the gene; and c) detectingthe expression of the green fluorescent protein in the cell, therebyindicating the expression of the gene in the cell.

This invention provides a method for indicating expression of a gene ina subject which comprises: a) introducing into a cell of the subject aDNA molecule having DNA sequence of the gene linked to DNA sequenceencoding a green fluorescent protein such that the regulatory element ofthe gene will control expression of the green fluorescent protein; b)culturing the cell in conditions permitting expression of the fusedprotein; and c) detecting the expression of the green fluorescentprotein in the cell, thereby indicating the expression of the gene inthe cell.

In an embodiment of the above methods, the green fluorescent protein isthe Aequorea victoria green fluorescent protein.

This invention provides a method for determining the tissue-specificityof transcription of a DNA sequence in a subject which comprises: a)introducing into a cell of the subject a DNA molecule having the DNAsequence linked to DNA sequence encoding a green fluorescent proteinsuch that the DNA sequence will control expression of the greenfluorescent protein; b) culturing the subject in conditions permittingthe expression of the green fluorescent protein; and c) detecting theexpression of the green fluorescent protein in different tissue of thesubject, thereby determining the tissue-specificity of the expression ofthe DNA sequence.

This invention provides a method for determining the presence of heavymetal in a solution which comprises: a) culturing the cell comprising aDNA molecule having a promoter from a gene, other than a greenfluorescent protein operatively linked to a DNA sequence encoding thegreen fluorescent protein, wherein transcription at the promoter isactivated by a heavy metal in the solution; and b) detecting expressionof the green fluorescent protein, the expression of the greenfluorescent protein indicates the presence of heavy metal.

This invention provides a method for detecting pollutants in a solutionwhich comprises: a) culturing the cell comprising a DNA molecule havinga promoter from a gene, other than a green fluorescent proteinoperatively linked to a DNA sequence encoding the green fluorescentprotein, wherein the promoter is activated by a heavy metal or a toxicorganic compound or the promoter is for a stress protein in thesolution; and b) detecting expression of the green fluorescent protein,the expression of the green fluorescent protein indicates the presenceof pollutants in the solution.

Finally, this invention provides a method for producing fluorescentmolecular weight markers comprising: a) linking a DNA molecule encodinga green fluorescent protein with a DNA molecule encoding a known aminoacid sequence in the same reading frame; b) introducing the linked DNAmolecule of step a) in an expression system permitting the expression ofa fluorescent protein encoded by the linked DNA molecule; and c)determining the molecular weight of the expressed fluorescent protein ofstep b), thereby producing a fluorescent molecular weight marker.

Various expression systems are known in the art. The E. coli expressionsystem, one of the commonly used system is described in the followingsection.

The determination of molecular weight may be done by comparing theexpressed fluorescent protein of step b) with known molecular weightmarkers. Alternatively, the molecular weight can be predicted bycalculation since the linked DNA sequence is known (and so is the aminoacid sequence being encoded). In an embodiment, the expressedfluorescent protein is purified. The purified fluorescent protein can beconveniently used as molecular weight markers.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

Experimental Details

A cDNA for the Aequorea victoria green fluorescent protein (GFP)produces a fluorescent product when expressed in prokaryotic(Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Becauseexogenous substrates and cofactors are not required for thisfluorescence, GFP expression can be used to monitor gene expression andprotein localization in living organisms.

Light is produced by the bioluminescent jellyfish Aequorea victoria whencalcium binds to the photoprotein aequorin (1). Although activation ofaequorin in vitro or in heterologous cells produces blue light, thejellyfish produces green light. This latter light is the result of asecond protein in A. victoria that derives its excitation energy fromaequorin (2), the green fluorescent protein (GFP).

Purified GFP, a protein of 238 amino acids (3), absorbs blue light(maximally at 395 nm with a minor peak at 470 nm) and emits green light(peak emission at 509 nm with a shoulder at 540 nm) (2, 4). Thisfluorescence is very stable; virtually no photobleaching is observed(5). Although the intact protein is needed for fluorescence, the sameabsorption spectral properties found in the denatured protein are foundin a hexapeptide that starts at amino acid 64 (6, 7). The GFPchromophore is derived from the primary amino acid sequence through thecyclization of Ser-dehydroTyr-Gly within this hexapeptide (7). Themechanisms that produce the dehydrotyrosine and cyclize the polypeptideto form the chromophore are unknown. To determine whether additionalfactors from A. victoria were needed for the production of thefluorescent protein, applicants tested GFP fluorescence in heterologoussystems. Here applicants show that UFP expressed in prokaryotic andeukaryotic cells is capable of producing a strong green fluorescencewhen excited by blue light. Because this fluorescence requires noadditional gene products from A. Victoria, chromophore formation is notspecies specific and occurs either through the use of ubiquitouscellular components or by autocatalysis.

Expression of GFP in Escherichia coli (8) under the control of the T7promoter results in a readily detected green fluorescence (9) that isnot observed in control bacteria. Upon illumination with a long-wave UVsource, fluorescent bacteria were detected on agar plates containing theinducer isopropyl-β-D-thiogalactoside (IPTG) (FIG. 1). When GFP waspartially purified from this strain (10), it was found to havefluorescence excitation and emission spectra indistinguishable fromthose of the purified native protein (FIG. 2). The spectral propertiesof the recombinant GFP suggest that the chromophore can form in theabsence of other A. victoria products.

Transformation of the nematode Caenorhabditis elegans also resulted inthe production of fluorescent GFP (11) (FIG. 3). GFP was expressed in asmall number of neurons under the control of a promoter for the mec-7gene. The mec-7 aene encodes a β-tubulin (12) that is abundant in sixtouch receptor neurons in C. elegans and less abundant in a few otherneurons (13, 14). The pattern of expression of GFP was similar to thatdetected by MEC-7 antibody or from mec-7lacZ fusions (13-15). Thestrongest fluorescence was seen in the cell bodies of the fourembryonically-derived touch receptor neurons (ALML, ALMR, PLML, PLMR) inyounger larvae. The processes from these cells, including their terminalbranches, were often visible in larval animals. In some newly hatchedanimals, the PLM processes were short and ended in what appeared to beprominent growth cones. In older larvae, the cell bodies of theremaining touch cells (AVM and PVM) were also seen; the processes ofthese cells were more difficult to detect. Thesepostembryonically-derived cells arise during the first of the fourlarval stages (16), but their outgrowth occurs in the following larvalstages (17), with the cells becoming functional during the fourth larvalstage (18). GFP's fluorescence in these cells is consistent with theseprevious results: no fluorescence was detected in these cells in newlyhatched or late first-stage larvae, but it was seen in four of ten latesecond-stage larvae, all nine early fourth-stage larvae, and seven ofeight young adults (19). In addition, moderate to weak fluorescence wasseen in a few other neurons (FIG. 3) (20). The details of the expressionpattern are being examined.

Like the native protein, GFP expressed in both E. coli and C. elegans isquite stable (lasting at least ten minutes) when illuminated with450-490 nm light. Some photobleaching occurs, however, when the cellsare illuminated with 340-390 nm or 395-440 nm light (21).

Several methods are available to monitor gene activity and proteindistribution within cells. These include the formation of fusionproteins with coding sequences for β-galactosidase, firefly luciferase,and bacterial luciferase (22). Because such methods requireexogenously-added substrates or cofactors, they are of limited use withliving tissue. Because the detection of intracellular GFP requires onlyirradiation by near UV or blue light, it is not substrate limited. Thus,it should provide an excellent means for monitoring gene expression andprotein localization in living cells (23, 24). Because it does notappear to interfere with cell growth and function, GFP should also be aconvenient indicator of transformation and one that could allow cells tobe separated using fluorescence-activated cell sorting. Applicants alsoenvision that GFP can be used as a vital marker so that cell growth (forexample, the elaboration of neuronal processes) and movement can befollowed in situ, especially in animals that are essentially transparentlike C. elegans and zebrafish. The relatively small size of the proteinmay facilitate its diffusion throughout the cytoplasm of extensivelybranched cells like neurons and glia. Since the GFP fluorescencepersists after treatment with formaldehyde (9), fixed preparations canalso be examined. In addition, absorption of appropriate laser light byGFP-expressing cells (as has been done for lucifer yellow-containingcells) (25), could result in the selective killing of the cells.

Further Experiments on GFP Expression

The TU#58 plasmid, which contains the green fluorescent protein (GFP)coding sequence in the pET3a expression vector (29) was transformed intoEscherichia coli strain BLR (DE3) (A. Roca, University of Wisconsin:cited in the Novogen Catalogue) using procedures described previously(29). The resulting strain (SMC3), because of the reduced recombinationof the host, was much more stable for GFP expression (all the colonieson plates with ampicillin but without the IPTG inducer (29) werebrightly fluorescent when viewed with a hand-held UV lamp).

A second construct (TU#147), similar to TU#58, was made with pET11 (A.H. Rosenberg, et al. 1987). Expression in BLR (DE3) from this plasmidwas more tightly controlled; expression was seen soon after IPTG wasadded, but only after some time without inducer.

The SMC3 strain was used to test the requirement for aerobic growth ofthe bacteria for the production of a fluorescent product. Plates weregrown under anaerobic conditions in a Gas-Pak container according to theinstructions of the manufacturer (Becton Dickinson MicrobiologySystems). Colony growth was slowed under anaerobic conditions and theresulting colones were not detectably fluorescent after at least 3 daysof growth under anaerobic conditions (using the hand-held UV lab).Colonies, however, became fluorescent after a day's exposure to room air(some fluorescence was seen after a few hours).

REFERENCES AND NOTES

1. O. Shimomura, F. H. Johnson, Y. Saiga, J. Cell. Comp. Physiol. 59,223 (1962).

2. J. G. Morin and J. W. Hastings, J. Cell. Physiol. 77, 313 (1971); H.Morise, O. Shimomura, F. H. Johnson, J. Winant, Biochemistry 13, 2656(1974).

3. D. C. Prasher, V. K. Eckenrode, W. W. Ward, F. G. Prendergast, M. J.Cormier, Gene 111, 229 (1992).

4. W. W. Ward, C. W. Cody, R. C. Hart, M. J. Cormier, Photochem.Photobiol. 31, 611 (1980).

5. F. G. Prendergast, personal communication.

6. O. Shimomura, FEBS Lett. 104, 220 (1979).

7. C. W. Cody, D. C. Prasher, W. M. Westler, F. G. Prendergast, W. W.Ward, Biochemistry 32, 1212 (1993).

8. Plasmid pGFP10.1 contains the EcoRI fragment encoding the GFP cDNAfrom λgfp10 (3) in pBS(+) (Stratagene®). The fragment was obtained byamplification with the polymerase chain reaction [PCR; R. K. Saiki etal., Science 239, 487 (1988)] with primers flanking the EcoRI sites andsubsequent digestion with EcoRI. DNA was prepared by the Magic Miniprepsprocedure (Promega) and sequenced (after an additional ethanolprecipitation) on an Applied Biosystems DNA Sequencer 370A at the DNASequencing facility at Columbia College of Physicians and Surgeons. Thesequence of the cDNA in pGFP10.1 differs from the published sequence bya change in codon 80 within the coding sequence from CAG to CGG, achange that replaces a glutamine residue with arginine [R. Heim, S. Emr,and R. Tsien (personal communication) first alerted us to a possiblesequence change in this clone and independently noted the same change.]This replacement has no detectable effect on the spectral properties ofthe protein (FIG. 2).

An E. coli expression construct was made with PCR to generate a fragmentwith an NheI site at the start of translation and an EcoRI site 5' tothe termination signal of the GFP coding sequence from PGFP10.1 . The 5'primer was ACAAAGGCTAGCAAAGGAGAAGAAC (Sequence ID No. 1) and the 3'primer was the T3 primer (Stratagene®). The NheI-EcoRI fragment wasligated into the similarly cut vector pET3a [A. H. Rosenberg et al.,Gene 56, 125 (1987)] by standard methods (26). The resulting codingsequence substitutes an Ala for the initial GFP Met, which becomes thesecond amino acid in the polypeptide. The E. coli strain BL21(DE3)Lys S[F. W. Studier and B. A. Moffat, J. Mol. Biol. 189, 113 (1986)] wastransformed with the resulting plasmid (TU#58) and grown at 37° C.Control bacteria were transformed with pET3a. Bacteria were grown onnutrient plates containing ampicillin (100 μg/ml) and 0.8 mM IPTG.Transformed bacteria from this transformation show green fluorescencewhen irradiated with ultraviolet light. A recombinant plasmid of thisbacteria was used for the experiments described here and the experimentin FIG. 2 and the experiment in Note 10. Several months later,applicants noticed that the bacterial colonies can be divided into twogroups: 1) strongly fluorescent; and 2) weakly fluorescent (applicantsbelieve that the weakly fluorescent may have mutated, disabled orpartial or completely deleted TU#58). One strongly fluorescent colonywas picked to generate the bacterial strain SMCl (ATCC Accession No.69554). [A similar PCR-generated fragment (see note 11) was used inapplicants' C. elegans construct. As others are beginning to usepGFP10.1, applicants have heard that while similar PCR fragments producea fluorescent product in other organisms (R. Heim, S. Emr, and R. Tsien,personal communication; S. Wang and T. Hazelrigg, personalcommunication; L. Lanini and F. McKeon, personal communication; see note23), the EcoRI fragment does not (R. Heim, S. Emr, and R. Tsien,personal communication; A. Coxon, J. R. Chaillet, and T. Bestor,personal communication). These results may indicate that elements at the5' end of the sequence or at the start of translation inhibitexpression.]

9. Applicants used a variety of microscopes (Zeiss Axiophot, NikonMicrophot FXA, and Olympus BH2-RFC and BX50) equipped forepifluorescence microscopy. Usually filter sets for fluoresceinisothiocyanate fluorescence were used (for example, the Zeiss filter setused a BP450-490 excitation filter, 510 nm dichroic, and either aBP515-565 or a LP520 emission filter), although for some experimentsfilter sets that excited at lower wavelengths were used (for example, aZeiss filter set with BP395-440 and LP470 filters and a 460 nm dichroicor with BP340-390 and LP400 filters with a 395 nm dichroic). In someinstances a xenon lamp appeared to give a more intense fluorescence thana mercury lamp when cells were illuminated with light around 470 nm,although usually the results were comparable. No other attempts weremade to enhance the signal (for example, by using low intensity lightcameras), although this may be useful in some instances.

Previous experiments had shown that the native protein was fluorescentafter glutaraldehyde fixation (W. W. Ward, unpub. data). S. Wang and T.Hazelrigg (personal communication; 23) have found that GFP fusionproteins in Drosophila melanogaster are fluorescent after formaldehydefixation. Applicants have confirmed that fluorescence persists afterformaldehyde fixation with applicants' C. elegans animals and withrecombinant GFP isolated from E. coli. The chemicals in nail polish,which is often used to seal cover slips, however, did appear tointerfere with the C. elegans GFP fluorescence.

10. In the applicants' initial experiments, GFP was purified from 250 mlcultures of BL21(DE3)Lys S bacteria containing TU#58; bacteria weregrown in LB broth (26) containing ampicillin (100 μg/ml) and 0.8 mMIPTG. Induction was best when IPTG was present continually.Nevertheless, subsequent experiments with bacterial strain SMC1 indicatethat the bacteria could not grow in the constant presence of IPTG butcan be induced by the IPTG during the log phase growth. The productionof fluorescent protein is best at room temperature. Cells were washed in4 ml of 10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM MgCl₂, and 10 mMdithiothreitol [A. Kumagai and W. G. Dunphy, Cell 64, 903 (1991)] andthen sonicated (2×20 sec) in 4 ml of the same buffer containing 0.1 mMPMSF, pepstatin A (1 μg/ml), leupeptin (1 μg/ml), and aprotinin (2μg/ml), and centrifuged at 5,000 rpm for 5 min in the cold. Thesupernatant was centrifuged a second time (15,000 rpm for 15 min) andthen diluted sevenfold with 10 mM Tris (pH 8.0), 10 mM EDTA, and 0.02%NaN₃. Corrected excitation and emission spectra were obtained with aSPEX FlT11 spectrofluorometer and compared with the purified Lisoprotein form of GFP from A. victoria (M. Cutler, A. Roth, and W. W.Ward, unpub. data). The excitation spectra were measured from 300-500 nmwith a fixed emission wavelength of 509 nm, and the emission spectrawere measured from 410-600 nm with a fixed excitation of 395 nm. Allspectra were recorded as signal-reference data (where the reference is adirect measurement of the lamp intensity with a separate photomultipliertube) at room temperature with 1 sec integration times and 1 nmincrements. The spectral band widths were adjusted to 0.94 nm for allspectra.

11. Wild-type and mutant C. elegans animals were grown and geneticstrains were constructed according to S. Brenner, Genetics 77, 71(1974).

The plasmid pGFP10.1 was used as a template for PCR (with the 5' primerGAATAAAAGCTAGCAAAGATGAGTAAAG (Sequence ID No. 2) and the 3' T3 primer)to generate a fragment with a 5' NheI site (at the start of translation)and a 3' EcoRI site (3' of the termination codon). The DNA was cut toproduce an NheI--EcoRI fragment that was ligated into plasmid pPD 16.51(12, 27), a vector containing the promoter of the C. elegans mec-7 gene.Wild-type C. elegans were transformed by coinjecting this DNA (TU#64)and the DNA for plasmid pRF4, which contains the dominant rol-6(sul006)mutation, into adult C. elegans gonads as described by C. M. Mello, J.M. Kramer, D. Stinchcomb, and V. Ambros, EMBO J. 10, 3959 (1991). Arelatively stable line was isolated (TU1710) and the DNA it carried wasintegrated as described by Mitani et al. (15) to produce the integratedelements uIs3 and uIs4 (in strains TU1754 and TU1755, respectively).

Living C. elegans animals were mounted on agar (or agarose) pads asdescribed (16), often with 10 mM NaN₃ as an anesthetic (28) (anothernematode anesthetic, phenoxypropanol, quenched the fluorescence) andexamined with either a Zeiss universal or axiophot microscope. For C.elegans, a long-pass emission filter works best because the animal'sintestinal autofluorescence, (which increases as the animal matures),appears yellow (with band-pass filters the autofluorescence appearsgreen and obscures the GFP fluorescence).

Because much more intense fluorescence was seen in uIs4 than uIs3animals (for example, it was often difficult to see the processes of theALM and PLM cells in uIs3 animals when the animals were illuminated witha mercury lamp), the former have been used for the observations reportedhere. The general pattern of cell body fluorescence was the same in bothstrains and in the parental, nonintegrated strain (fluorescence in thisstrain was as strong as that in the uIs4 animals) . The uIs4 animals,however, did show an unusual phenotype: both the ALM and PLM touch cellswere often displaced anteriorly. The mature cells usually had processesin the correct positions, although occasional cells hadabnormally-projecting processes. These cells could be identified astouch receptor cells, because the fluorescence was dependent on mec-3, ahomeobox gene that specifies touch cell fate (13, 15, 18, 28). mec-7expression is reduced in the ALM touch cells of the head (but not asdramatically in the PLM touch cells of the tail) in mec-3 gene mutants(13, 15). Applicants find a similar change of GFP expression in a mec-3mutant background for both uIs3 and uIs4. Thus, GFP accuratelyrepresents the expression pattern of the mec-7 gene. It is likely thatthe reduced staining in uIs3 animals and the misplaced cells in uIs4animals is the result of either secondary mutations or the amount andposition of the integrated DNA.

12. C. Savage, M. Hamelin, J. G. Culotti, A. Coulson, D. G. Albertson,M. Chalfie, Genes Dev. 3, 870 (1989).

13. M. Hamelin, I. M. Scott, J. C. Way, J. G. Culotti, EMBO J. 11, 2885(1992).

14. A. Duggan and M. Chalfie, unpub. data.

15. S. Mitani, H. P. Du, D. H. Hall, M. Driscoll, M. Chalfie,Development 119, 773 (1993).

16. J. E. Sulston and H. R. Horvitz, Develop. Biol. 56, 110 (1977).

17. W. W. Walthall and M. Chalfie, Science 239, 643 (1988).

18. M. Chalfie and J. Sulston, Dev. Biol. 82, 358 (1981).

19. In adults, the thicker size of the animals and the more intenseautofluorescence of the intestine tend to obscure these cells.

20. These include several cells in the head (including the FLP cells)and tail of newly hatched animals and the BDU cells, a pair of neuronsjust posterior to the pharynx. Expression of mec-7 in these cells hasbeen seen previously (13, 15). The strongest staining of these non-touchreceptor neurons are a pair of cells in the tail that have anteriorlydirected processes that project along the dorsal muscle line. It islikely that these are the ALN cells, the sister cells to the PLM touchcells [J. G. White, E. Southgate, J. N. Thomson, and S. Brenner, Philos.Trans. R. Soc. Lond. B Biol. Sci. 314, 1 (1986).]

21. The photobleaching with 395-440 nm light is further accelerated, towithin seconds, in the presence of 10 mM NaN₃, which is used as a C.elegans anesthetic (11). However, when cells in C. elegans have beenphotobleached, some recovery is seen within 10 min. Furtherinvestigation is needed to determine whether this recovery represents denovo synthesis of GFP. Rapid photobleaching (complete within a minute)of the green product was also seen when C. elegans was illuminated with340-390 nm light. Unlike the photobleaching with 395-440 nm light, whichabolished fluorescence produced by the 340-390 or 450-490 nm light,photobleaching with 340-390 nm light did not appear to affect thefluorescence produced by 395-490 or 450-490 nm light. Indeed, thefluorescence produced by 450-490 nm light appeared to be more intenseafter brief photobleaching by 340-390 nm light. This selectivephotobleaching may indicate the production of more than one fluorescentproduct in the animal. These data on GFP fluorescence within E. coli andC. elegans is in contrast to preliminary studies that suggest that theisolated native and E. coli proteins are very photostable. Applicants donot know whether this in vivo sensitivity to photobleaching is a normalfeature of the jellyfish protein (the fluorescence in A. victoria hasnot been examined) or results from the absence of a necessaryposttranslational modification unique to A. victoria or nonspecificdamage within the cells.

22. Reviewed in T. J. Silhavy and J. R. Beckwith, Microbiol. Rev. 49,398 (1985); S. J. Gould and S. Subramani, Anal. Biochem. 175, 5 (1988);and G. S. A. B. Stewart and P. Williams, J. Gen. Microbiol. 138, 1289(1992).

23. R. Heim, S. Emr, and R. Tsien (personal communication) have foundthat GFP expression in Saccharomyces cerevisiae can make the cellsstrongly fluorescent without causing toxicity. S. Wang and T. Hazelrigg(personal communication) have found that both C-terminal and N-terminalprotein fusions with GFP are fluorescent in Drosophila melanogaster. L.Lanini and F. McKeon (personal communication) have expressed a GFPprotein fusion in mammalian (COS) cells. E. Macagno (personalcommunication) is expressing GFP in leeches. T. Hughes (personalcommunication) is expressing GFP in mammalian HEK293 cells.

24. Applicants have generated several other plasmid constructions thatmay be useful to investigators. These include a pBluescript II KS (+)derivative (TU#65) containing a KpnI--EcoRI fragment encoding GFP withan AgeI site 5' to the translation start and a BsmI site at thetermination codon. Also available are gfp versions (TU#60-TU#63) of thefour C. elegans lacZ expression vectors (pPDl6.43, pPD21.28, pPD22.04,and pPD22.11, respectively) described by Fire et al., 1990 (27) exceptthat they lack the KpnI fragment containing the SV40 nuclearlocalization signal.

25. J. P. Miller and A. Selverston, Science 206, 702 (1979).

26. J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular cloning: Alaboratory manual, 2nd Ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, New York, (1989).

27. A. Fire, S. W. Harrison, and D. Dixon, Gene 93, 189 (1990).

28. J. C. Way and M. Chalfie, Cell 54, 5 (1988).

29. Chalfie, M., Tu. Y., Euskirchen, G., Ward, W. W., Prasher, D. C.,Science 263, 802 (1994).

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 2                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: NO                                                   -     (vi) ORIGINAL SOURCE:                                                   #coli     (A) ORGANISM: Escherichia                                           -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #               25 GAGA AGAAC                                                 - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 28 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: NO                                                   -     (vi) ORIGINAL SOURCE:                                                   #coli     (A) ORGANISM: Escherichia                                           -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #             28   AGAT GAGTAAAG                                              __________________________________________________________________________

What is claimed is:
 1. A cell comprising a DNA molecule having aregulatory element from a gene, other than a gene encoding a greenfluorescent protein, operatively linked to a DNA sequence encoding afluorescent mutant of green fluorescent protein of Aequorea victoria. 2.The cell of claim 1, wherein the cell is selected from the groupconsisting of a bacterial cell, a yeast cell, a fungal cell, a plantcell and an animal cell.
 3. The cell of claim 1, wherein the regulatoryelement is a promoter.
 4. The cell of claim 1, wherein the regulatoryelement is an enhancer.
 5. The cell of claim 1, wherein the cell is anE. coli cell.
 6. A method to produce a fluorescent mutant of greenfluorescent protein of Aequorea victoria comprising:a) culturing thecell of claim 1 so that the cell produces the fluorescent mutant; and b)isolating and purifying the fluorescent mutant so produced by the cell.7. The cell of claim 1, wherein the cell is an insect cell or a nematodecell.
 8. The cell of claim 3, wherein the promoter is activated by aheavy metal.
 9. The cell of claim 3, wherein the promoter is that from aP450 gene.
 10. The cell of claim 3, wherein the promoter is from a geneencoding a stress protein.
 11. The cell of claim 3, wherein the promoteris from a gene required for cell viability.
 12. The cell of claim 5designated pGFP10.1 (ATCC Accession No. 75547).
 13. The method of claim6, wherein the cell is an E. coli cell.
 14. The cell of claim 10,wherein the stress protein is a heat-shock protein.
 15. The method ofclaim 13, wherein the cell is cultured aerobically.
 16. A method forselecting cells expressing a protein of interest which comprises:a)introducing into the cells a first DNA molecule having a DNA sequenceencoding the protein of interest and a second DNA molecule having a DNAsequence encoding a fluorescent mutant of green fluorescent protein ofAeguorea victoria; b) culturing cells resulting from step (a) underconditions permitting expression of the fluorescent mutant of greenfluorescent protein and the protein of interest; and c) selecting thecultured cells which express the fluorescent mutant of green fluorescentprotein, thereby selecting cells expressing the protein of interest. 17.The method of claim 16, wherein the first DNA molecule and the secondDNA molecule are linked.
 18. The method of claim 16, wherein the cellsare selected from the group consisting of bacterial cells, yeast cells,fungal cells, plant cells and animal cells.
 19. The method of claim 16,wherein the cells are insect cells or nematode cells.
 20. A method forlocalizing a protein of interest in a cell which comprises:a)introducing into a cell a DNA molecule having a sequence encoding theprotein of interest linked to a DNA sequence encoding a fluorescentmutant of green fluorescent protein of Aequorea victoria such that theprotein produced by the DNA molecule will have the protein of interestfused to the fluorescent mutant of green fluorescent protein of Aequoreavictoria; b) culturing the cell under conditions permitting expressionof the fused protein; and c) detecting the location of the fusedprotein, thereby localizing the protein of interest in the cell.
 21. Themethod of claim 20, wherein the cell normally expresses the protein ofinterest.
 22. A method of detecting expression of a gene in a cell whichcomprises:a) introducing into the cell a DNA molecule having thesequence of the gene linked to a DNA molecule encoding a fluorescentmutant of green fluorescent protein of Aequorea victoria such that aregulatory element of the gene controls expression of the fluorescentmutant of green fluorescent protein of Aequorea victoria; b) culturingthe cell under conditions permitting expression of the gene; and c)detecting the expression of the fluorescent mutant of green fluorescentprotein of Aequorea victoria in the cell, thereby detecting expressionof the gene in the cell.
 23. A method for detecting expression of a genein a living organism which comprises:a) introducing into a cell of thesubject a DNA molecule having the sequence of the gene linked to a DNAmolecule encoding a fluorescent mutant of green fluorescent protein ofAequorea victoria such that a regulatory element of the gene controlsexpression of the fluorescent mutant of green fluorescent protein ofAequorea victoria; b) culturing the cell under conditions permittingexpression of the gene; and c) detecting the expression of thefluorescent mutant of green fluorescent protein of Aequorea victoria inthe cell, thereby detecting expression of the gene in the livingorganism.
 24. A method for determining the tissue-specificity oftranscription of a first DNA sequence in a living organism whichcomprises:a) introducing into a cell of the living organism a DNAmolecule which comprises the first DNA sequence linked to a second DNAsequence encoding a fluorescent mutant of green fluorescent protein ofAequorea victoria such that the first DNA sequence controls expressionof the fluorescent mutant of green fluorescent protein of Aequoreavictoria in the living organism; and detecting expression of thefluorescent mutant of green fluorescent protein of Aequorea victoria indifferent tissues of the living organism, thereby determining thetissue-specificity of the transcription of the first DNA sequence in theliving organism.
 25. A method for producing a fluorescent molecularweight protein marker comprising:a) linking a first DNA moleculeencoding a fluorescent mutant of green fluorescent protein of Aequoreavictoria with a second DNA molecule encoding a known amino acid sequencewhich is in the same reading frame as the first DNA molecule; b)introducing the linked DNA molecule of step (a) into a proteinexpression system permitting the expression of a fusion proteincomprising the fluorescent mutant linked to the known amino acidsequence; c) recovering the fusion protein expressed in step (b): and d)determining the molecular weight of the fusion protein from step (c),thereby producing a fluorescent molecular weight protein marker.
 26. Themethod of claim 25, further comprising purification of the expressedprotein.